therascreen BRAF Pyro Kit

For sequencing-based detection and quantitation of cancer-related gene mutations in the BRAF gene

Compliance with EU IVD Directive 98/79/EC

Comprehensive results in real time

Accurate quantitation of cancer-related BRAF gene mutations

Easy interpretation of complex sequence information

The therascreen BRAF Pyro Kit is a molecular detection kit for the identification of mutations in the BRAF gene. The kit contains primers and reagents for amplification of the BRAF gene plus buffers, primers, and reagents for detection and quantification of mutations in real time using Pyrosequencing technology on the PyroMark Q24 System.

The therascreen BRAF Pyro Kit is intended for in vitro diagnostic use.

Linearity of V600E mutation.

Linearity for the mutation V600E (GTG->GAG) in codon 600.

Performance

Linearity

Linearity was determined using mixtures of plasmids carrying the wild type or mutant
sequence for the mutation V600E (GTG->GAG) in codon 600 of the BRAF gene (see
figure "Linearity
of V600E mutation"). The plasmids were mixed in proportions to give four
levels of mutation (5, 10, 30, and 50%). Each mixture was analyzed with three different
lots of the therascreen BRAF Pyro Kit in three Pyrosequencing runs with
three replicates each.

The results were linear within an allowable nonlinearity of 5 % units in the tested
range of 5 to 50% mutation level.

Precision

The precision data allows the determination of the total variability of the assays
and was obtained at three different levels by analysis of the above mentioned plasmid
mixtures with three replicates each.

Repeatability (intra-assay and inter-batch variability) was calculated based on
the data for determination of linearity (three runs on the same day using varying
lots of the therascreen BRAF Pyro Kit). Intermediate precision (intra-laboratory
variability) was determined in three runs within one laboratory on three different
days with varying operators, PyroMark Q24 systems, and lots of the therascreen
BRAF Pyro Kit. Reproducibility (inter-laboratory variability) was calculated from
two runs each in an internal and external laboratory and using varying lots of the
therascreen BRAF Pyro Kit.

Precision estimates are expressed as standard deviation of the measured mutation
frequencies in % units. The repeatability, intermediate precision, and reproducibility
for the mutation V600E (GTG->GAG) in codon 600 was 0.6–2.1, 0.7–1.8, and 0.8–2.1
% units, respectively, in the measured range of 5 to 50% mutation level.

Precision values for the mutation V600E (GTG->GAG) in codon 600

% mutated plasmid

Repeatability (Mean, SD)

Intermediate precision (Mean, SD)

Reproducibility (Mean, SD)

5

5.2, 0.6

4.4, 0.7

5.1, 0.8

10

9.1, 1.0

9.6, 1.0

9.6, 1.3

30

28.1, 2.1

27.9, 1.8

28.3, 2.1

50

46.9, 1.2

46.3, 1.5

47.9, 1.7

All values are given as % units. SD: standard deviation (n=9). % mutated plasmid
based on OD260 measurement.

Principle

The therascreen BRAF Pyro Kit is used for quantitative measurements of mutations in codons 600 and 464–469 of the human BRAF gene in real time using Pyrosequencing technology on the PyroMark Q24 System. The BRAF gene encodes the kinase v-raf murine sarcoma viral oncogene homolog B1, a proto-oncogene that acts downstream of EGFR. Mutations in the BRAF gene are found in 6–8% of all cancers and more frequently in melanoma (50–60%), colorectal cancer (10%), and thyroid (39%) cancers. Approximately 90% of these mutations are V600E substitutions.

The following mutations are detected:

Codon 600: V600A, V600E, V600G, and V600M

Codon 464–469: G464E, G464V, G466E, G466V, G469A, G469E, and G469V

Procedure

The product consists of 2 assays: one for detecting mutations in codon 600 and the other for detecting mutations in codons 464–469 (see figure "Illustration of the BRAF assay"). The two regions are amplified separately by PCR and sequenced through the defined region. Sequences surrounding the defined positions serve as normalization and reference peaks for quantification and quality assessment of the analysis.

The therascreen BRAF Pyro Kit is intended to be used as an aide to identify melanoma or colorectal cancer patients more likely to benefit from treatment with cancer therapies that are selective inhibitors of BRAF. The kit is used for the detection of mutations in codons 600 and 464–469 of the human BRAF gene.